Tobacco sesquiterpene synthase nttps122 and application thereof

Abstract

The application belongs to the technical field of plant molecular biology and plant genetic engineering, and particularly relates to a tobacco sesquiterpene synthase NtTPS122 and application thereof. The amino acid sequence of the tobacco sesquiterpene synthase NtTPS122 is shown as SEQ ID NO. 1. The application finds that NtTPS122 can be expressed in large quantities through E. coli, and can efficiently catalyze farnesyl pyrophosphate to generate beta-Longipinene. The application lays a foundation for research on tobacco terpene secondary metabolism, provides important candidate genes for carrying out tobacco genetic engineering research and stress resistance breeding, and has important significance for producing beta-Longipinene by using genetic engineering technology.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and plant genetic engineering technology, specifically relating to a tobacco sesquiterpene synthase NtTPS122 and its applications. Background Technology

[0002] Terpenes are natural products that are widely distributed in nature and have the most diverse chemical structures. Volatile terpenes possess unique odors and biological activities, and play important biological functions in plant growth and development, as well as in insect and disease resistance. Based on the number of isoprene units (C5) in their structure, terpenes are classified into monoterpenes (C6, C7, C8, C9 ... 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), triterpenes (C 30 In plant cells, the precursor compound geranyl pyrophosphate (GPP, C...) is synthesized via the mevalonate pathway (MVA pathway) in the cytoplasm and the methyl erythritol-4-phosphate pathway (MEP pathway) in the plastids. 10 ), farnesyl pyrophosphate (FPS, C 15 ) and geraniol geraniol pyrophosphate (GGPP, C 20 Terpene synthases (TPS) catalyze the formation of monoterpenes, sesquiterpenes, and diterpenes with diverse structures, which are then directly released into the environment or stored in specific organs, tissues, or cells. Alternatively, they undergo oxidation, reduction, and glycosylation modifications by cytochrome P450, alcohol dehydrogenases, and glycoside transferases to form structurally diverse derivatives. While the MVA and MEP pathways are relatively conserved in higher plants, TPS exhibits diverse catalytic activities and expression characteristics, determining the structural diversity, distribution characteristics, and environmental response patterns of plant terpenes.

[0003] β-Longipinene (molecular formula C) 15 H 24 β-Longipinene (molecular weight 204.35, CAS number 5989-08-2) is a rare sesquiterpene compound, detected only in *Scapania undulata* (Phytochemistry, 1977, Vol. 16, 1731-1751), and in extremely low amounts. Therefore, our understanding of the chemical properties and physiological functions of β-Longipinene is currently limited, restricting its development and utilization. Isolating and identifying the TPS gene capable of synthesizing β-Longipinene is of great significance for the efficient production of β-Longipinene through synthetic biology methods, for studying its functions in medicine, fragrances, cosmetics, and other fields, and for its industrial development and application. Summary of the Invention

[0004] This invention has discovered a sesquiterpene synthase derived from tobacco that can be expressed in large quantities using genetically engineered Escherichia coli, and this enzyme has a good catalytic effect on the synthesis of β-Longipinene.

[0005] In a first aspect, the present invention provides a tobacco sesquiterpene synthase NtTPS122, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] A second aspect of the invention provides the use of the tobacco sesquiterpene synthase NtTPS122 in the catalytic synthesis of β-Longipinene.

[0007] Furthermore, the substrate is farnesyl pyrophosphate.

[0008] A third aspect of the invention provides a gene for encoding the tobacco sesquiterpene synthase NtTPS122.

[0009] Furthermore, the gene has a nucleotide sequence as shown in any one of a1-a2:

[0010] a1, the nucleotide sequence shown in SEQ ID NO.2;

[0011] a2, encoding the tobacco sesquiterpene synthase NtTPS122, but with a different nucleotide sequence from that shown in SEQ ID NO.2 due to the degeneracy of the genetic code.

[0012] A fourth aspect of the invention provides a recombinant expression vector comprising the said gene.

[0013] Furthermore, the recombinant expression vector is obtained by inserting the gene forward between the BamHI and SacI sites of the pET28a plasmid.

[0014] A fifth aspect of the invention provides a genetically engineered bacterium comprising the recombinant expression vector.

[0015] Furthermore, the host bacterium used in the genetically engineered bacteria is Escherichia coli.

[0016] A fifth aspect of the present invention provides a method for preparing β-Longipinene, using the aforementioned tobacco sesquiterpene synthase NtTPS122 as a catalyst, farnesyl pyrophosphate as a substrate, and catalytically synthesizing β-Longipinene in the presence of dithiothreitol (DTT) and MgCl2.

[0017] The present invention has the following beneficial effects:

[0018] This invention expresses the tobacco-derived sesquiterpene synthase NtTPS122 in Escherichia coli and verifies its function in β-Longipinene synthesis. It was found that NtTPS122 can be expressed in large quantities in Escherichia coli and can efficiently catalyze the production of β-Longipinene from farnesyl pyrophosphate.

[0019] This invention combines the prokaryotic soluble expression of NtTPS122 with its specific and efficient catalytic activity towards the target product, significantly exceeding the level of existing technologies and possessing broad prospects for industrial application and potential for large-scale development. Attached Figure Description

[0020] Figure 1 Total ion chromatogram (TIC) for GC-MS analysis of the in vitro catalytic activity of recombinant NtTPS122 protein. The x-axis represents retention time (min), and the y-axis represents peak height of the chromatographic response.

[0021] Figure 2 This is a comparison of mass spectra of sesquiterpenes synthesized using NtTPS122 catalysis and β-Longipinene. The horizontal axis represents the mass spectrum of the NtTPS122-catalyzed product, and the horizontal axis below represents the mass spectrum of β-Longipinene.

[0022] Figure 3 This is the mass spectrum of the sesquiterpene product β-Longipinene. The horizontal axis represents the mass-to-ion charge ratio, and the vertical axis represents the ion fragment intensity.

[0023] Figure 4 The expression pattern of NtTPS122 in various tissues of tobacco. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1: Cloning of the tobacco NtTPS122 gene

[0026] Grind 100 mg of tobacco material thoroughly in liquid nitrogen, transfer to a 1.5 mL centrifuge tube, add 1 mL of Trizol (Invitrogen, Cat. 15596-018), mix well, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 10 min, and discard the precipitate. Add 0.2 mL of chloroform to the supernatant, mix well, centrifuge at 12000 rpm for 10 min, and add 0.5 mL of isopropanol to the supernatant to precipitate RNA. Centrifuge at 12000 rpm for 10 min, and dissolve the precipitate in 100 μL of water.

[0027] Reverse transcription was performed using an RNA PCR system (TaKaRa, Cat DRR019A). The reaction mixture consisted of: 1 μL 10× buffer, 1 μL dNTPs, 2 μL MgCl2, 1 μL Oligo dT, 0.5 μL RNA inhibitor, 0.5 μL AMV RTase, and 2 μL RNA. The reaction was incubated at 42°C for 30 min, followed by an ice bath.

[0028] PCR amplification was performed using cDNA obtained from reverse transcription as a template. The forward primer was NtTPS122-F: 5'-ATGGCCGCAGCAGCAGTTGG-3' (SEQ ID NO.3), and the reverse primer was NtTPS122-R: 5'-TCAAATTTCGATGGAGTCCAC-3' (SEQ ID NO.4). PCR amplification was performed using TakaRa PrimeSTAR Max DNA polymerase. The reaction mixture consisted of: 5 μL 10×Buffer, 2 μL 10 mM dNTPs, 2 μL forward primer, 2 μL reverse primer, 1 μL DNA polymerase, and 1 μL cDNA. The PCR conditions were: 95℃ for 5 min; 35 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 120 s; and a final extension at 72℃ for 5 min. The PCR products were detected by 1% agarose gel electrophoresis. The size of the NtTPS122 gene fragment was 1647 bp, which was consistent with the expected size.

[0029] The nucleotide sequence of the NtTPS122 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.1. Sequence homology alignment analysis results indicate that this protein is a typical sesquiterpene synthase.

[0030] Example 2: Construction of expression vector and transformation of E. coli

[0031] The target gene fragment was recovered using an agarose gel electrophoresis recovery kit and then ligated into a vector. The cloning vector (TransGold: CB101-01) was then transformed into *E. coli* DH5α clone strains. The transformation conditions were as follows: 5 μL of ligation product was added to 100 μL of competent cells, gently mixed, and incubated on ice for 30 min; the cells were then rapidly heat-shocked in a 42°C water bath for 90 s, immediately placed on ice for 2-3 min; 800 μL of LB broth was added, and the cells were incubated at 37°C with gentle shaking for 1 h; the bacterial suspension was centrifuged at 6000 rpm for 1 min, 700 μL of supernatant was discarded, the bacterial cells were resuspended, and plated onto LB agar plates containing ampicillin (Amp, 100 mg / L), and incubated upside down in the dark for 12–16 h. Positive clones were screened using colony PCR, and positive single colonies were selected. Plasmids were extracted and sent for sequencing.

[0032] Sequencing analysis revealed the cloning of the tobacco sesquiterpene synthase gene NtTPS122, whose nucleotide sequence is shown in SEQ ID NO.2. It contains 1647 bases and encodes a protein named sesquiterpene synthase NtTPS122, consisting of 548 amino acids, the specific amino acid sequence of which is shown in SEQ ID NO.1. Therefore, the insertion of the sesquiterpene synthase gene NtTPS122 into... The recombinant plasmid of the cloning vector was named pEASY-TPS122.

[0033] Using pEASY-TPS122 plasmid as a template, the NtTPS122 gene was amplified using TakaRa PrimeSTAR Max DNA polymerase. The primers were: ORF-NtTPS122-F-BamHI: 5'-GCGGATCCATGGCCGCAGCAGCAGTTGG-3' (SEQ ID NO.5) and ORF-NtTPS122-R-SacI: 5'-GCGAGCTCTCAAATTTCGATGGAGTCCA-3' (SEQ ID NO.6). The reaction mixture consisted of: 5 μL 10×Buffer, 2 μL 10 mM dNTPs, 2 μL forward primer, 2 μL reverse primer, 1 μL DNA polymerase, and 1 μL cDNA. PCR conditions were: 95℃ for 5 min; 35 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 120 s; and a final extension at 72℃ for 5 min. PCR products were detected by 1% agarose gel electrophoresis. The target gene fragment was recovered using an agarose gel recovery kit, double-digested with BamHI and SacI, and ligated to the double-digested pET28a plasmid (Novagen: 69864-3). The ligation product was transformed into E. coli, clones were picked and cultured, and plasmids were extracted and sequenced. The positive plasmid was named pET28-TPS122.

[0034] Example 3: Prokaryotic Expression and Activity Assay

[0035] pET28-TPS122 was transformed into *E. coli* Rosetta (DE3). Single colonies were picked and inoculated into liquid LB medium containing 50 mg / L kanamycin and cultured overnight. The culture was expanded to 50 ml until OD600 = 0.6, and IPTG (isopropyl β-D-thiogalactoside) was added to a final concentration of 1 mM. Induction culture was continued at 20°C for 12 hours. The bacterial culture was centrifuged at 1000 rpm for 1 min, and the pellet was resuspended in lysis buffer (50 mM Tris, pH 7.5, 5 mM DTT, 5 mM MgCl2). Cells were sonicated and centrifuged at 10000 rpm for 2 min. The supernatant was collected for cell viability assay.

[0036] Add 2 μL of FPP (Sigma-Aldrich, F6892) to 100 μL of the above protein, react at 37℃ for 1 hour, add 1 ml of n-hexane and shake, then take 1 μL for gas chromatography-mass spectrometry (GC-MS) analysis. GC instrument model: Thermo Trace 1300 (HP-5ms: 30m × 0.25mm × 0.25μm). Mass spectrometer model: Thermo ITQ 900 (EI ion source; ion trap detector). Injection volume: 1 μL. Chromatographic conditions: 60℃ for 3 min, ramp to 280℃ at 10℃ / min, hold for 5 min, helium flow rate 1 ml / min. Mass spectrometry conditions: ion source temperature 250℃, interface temperature 250℃, scan mode acquisition m / z 50-500.

[0037] GC-MS detection results showed that a product peak was detected at 15.3 minutes. Figure 1 The mass spectra were compared with those of the NIST library, and the results showed that the compound was β-Longipinene. Figure 2-3 ).

[0038] Example 4: Expression pattern of NtTPS122 gene

[0039] Grind 100 mg of tobacco material thoroughly in liquid nitrogen, transfer to a 1.5 mL centrifuge tube, add 1 mL of Trizol (Invitrogen, Cat. 15596-018), mix well, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 10 min, and discard the precipitate. Add 0.2 mL of chloroform to the supernatant, mix well, centrifuge at 12000 rpm for 10 min, and add 0.5 mL of isopropanol to the supernatant to precipitate RNA. Centrifuge at 12000 rpm for 10 min, and dissolve the precipitate in 100 μL of water.

[0040] Reverse transcription was performed using an RNA PCR system (TaKaRa, Cat DRR019A). The reaction mixture consisted of: 1 μL 10× buffer, 1 μL dNTPs, 2 μL MgCl2, 1 μL Oligo dT, 0.5 μL RNA inhibitor, 0.5 μL AMV RTase, and 2 μL RNA. The reaction was incubated at 42°C for 30 min, followed by an ice bath.

[0041] Using cDNA obtained from reverse transcription as a template, the expression level of NtTPS122 was detected using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The forward primer was qRT-NtTPS122-F: 5'-TGAACGTCTTGGCATCTCCTACC-3' (SEQ ID NO.7), and the reverse primer was qRT-NtTPS122-R: 5'-CGTCAGCATGAGTCCTTACATGTG-3' (SEQ ID NO.8). Tobacco EF-1a was used as an internal reference gene, and the primers were qRT-EF-1a-F: 5'-TGAGATGCACCACGAAGCTC-3' (SEQ ID NO.9) and qRT-EF-1a-R: 5'-CCAACATTGTCACCAGGAAGTG-3' (SEQ ID NO.10). The detection was performed using ChamQ SYBR qPCR Master Mix (Novizan). The PCR reaction system consisted of: 10 μL of 2×ChamQ SYBR qPCR Master Mix, 1 μL of forward primer, 1 μL of reverse primer, and 0.5 μL of cDNA. The PCR conditions were: 95℃ for 5 min; 95℃ for 15 s, 58℃ for 30 s, and 72℃ for 30 s; for 40 cycles.

[0042] The results showed that NtTPS122 was expressed at a high level in flowers, but not detected in roots, stems, leaves, flower buds, and other organs. Figure 4 ).

[0043] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0044] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a tobacco sesquiterpene synthase NtTPS122 in the catalytic synthesis of β-Longipinene, characterized in that, The amino acid sequence of the tobacco sesquiterpene synthase NtTPS122 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the tobacco sesquiterpene synthase NtTPS122 is shown in SEQ ID NO.2; the substrate is farnesyl pyrophosphate.

2. A method for preparing β-Longipinene, characterized in that, Using the tobacco sesquiterpene synthase NtTPS122 as described in claim 1 as a catalyst, and farnesyl pyrophosphate as a substrate, β-Longipinene was synthesized by catalysis in the presence of dithiothreitol and MgCl2.

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